Serological methods and diagnostic tests for syphilis antibodies

ABSTRACT

A method of detecting antibodies to syphilis antigens includes providing a syphilis detection kit having a screening solid-phase platform with a screening portion coated with non-treponemal syphilis antigens. The screening portion is fabricated by initially dissolving cholesterol in an organic solvent and further diluting the dissolved cholesterol in an ethanol solution comprising cardiolipin and lecithin to form an antigen solution, permitting the antigen solution to evaporate at the screening portion and at least partially coating the screening portion with an antigen coating, and stabilizing the antigen coating into a syphilis antigen complex by overcoating the antigen coating with an overcoat solution comprising an inert protein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is:

-   -   a continuation of copending U.S. patent application Ser. No.        14/561,333, filed, Dec. 5, 2014; and    -   a continuation of U.S. patent application Ser. No. 14/191,014,        filed Feb. 26, 2014, now U.S. Pat. No. 8,945,827 (which        application claims the priority, under 35 U.S.C. §119, of U.S.        Provisional Patent Application No. 61/778,544, filed Mar. 13,        2013),        of which priority is claimed under 35 U.S.C. §120; the prior        applications are herewith incorporated by reference herein in        their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

FIELD OF THE INVENTION

The present invention lies in the field of medical testing. The presentdisclosure relates to serological methods and diagnostic tests forsyphilis antibodies.

BACKGROUND OF THE INVENTION

Syphilis is a sexually transmitted infection caused by the spirochetebacterium treponema pallidum subspecies pallidum. The primary route oftransmission is through sexual contact; it may also be transmitted frommother to fetus during pregnancy or at birth, resulting in congenitalsyphilis. Other human diseases caused by related treponema palliduminclude yaws (subspecies pertenue), pinta (subspecies carateum), andbejel (subspecies endemicum).

The signs and symptoms of syphilis vary depending in which of the fourstages it presents (primary, secondary, latent, and tertiary). Theprimary stage classically presents with a single chancre (a firm,painless, non-itchy skin ulceration), secondary syphilis with a diffuserash which frequently involves the palms of the hands and soles of thefeet, latent syphilis with little to no symptoms, and tertiary syphiliswith gummas, neurological, or cardiac symptoms. It has, however, beenknown as “the great imitator” due to its frequent atypicalpresentations. Diagnosis is usually via blood tests; however, thebacteria can also be detected using dark field microscopy. Syphilis canbe effectively treated with antibiotics, specifically the preferredintramuscular penicillin G (given intravenously for neurosyphilis), orelse ceftriaxone, and in those who have a severe penicillin allergy,oral doxycycline, or azithromycin.

Syphilis is believed to have infected 12 million people worldwide in1999, with greater than 90% of cases in the developing world. Afterdecreasing dramatically since the widespread availability of penicillinin the 1940s, rates of infection have increased since the turn of themillennium in many countries, often in combination with humanimmunodeficiency virus (HIV). This has been attributed partly to unsafesexual practices among men who have sex with men, increased promiscuity,prostitution, and decreasing use of barrier protection.

As set forth above, diagnosis of syphilis is usually through bloodtests. There are presently two types of serological tests used for thediagnosis and treatment of syphilis infection: treponemal tests, whichutilize antigens prepared from the causative agent treponema pallidum,and non-treponemal tests, which employ antigens not derived directlyfrom the causative agent.

The Centers for Disease Control (CDC), Atlanta, Ga., recommends thatsyphilis screening, and the assessment of treatment, be performed firstusing non-treponemal tests because it is believed that non-treponemaltests are more sensitive. In such tests, a false positive is possible.The CDC further recommends that positive screening test results beconfirmed with a treponemal test. It is noted that the non-treponemaltests can be used for monitoring post-treatment and for detection ofre-infection. There are currently two non-treponemal tests in commonusage: the Venereal Disease Research Laboratory (VDRL) Test and theRapid Plasma Reagin (RPR) Test.

The VDRL Test is a non-treponemal serological screening for syphilisused to assess response to therapy, to detect CNS involvement, and as anaid in the diagnosis of congenital syphilis. The basis of the test isthat antibody (IgG, IgM or IgA), produced by a patient with syphilis,reacts with a compound comprised of cardiolipin, cholesterol, andlecithin. The test is performed by mixing the patients serum withcompound described above. Positive sera result in “flocculation.” Simplyput, the VDRL Test is a manual coagulation test with a qualitative,subjective, visual readout requiring the use of a microscope. Morespecifically, a sample of the patient's antibody is mixed in a testtube, or on a microscope slide. A clinician is required to view the testtube, or slide and determine, based upon the clinician's own experienceand training, if the visual presentation indicates coagulation to anextent sufficient to call the results positive. Quantitative testresults require a serial dilution of the test sample and testing ofmultiple dilutions. Thus, the VDRL Test is labor intensive and unable tobe automated. It is known that false negatives arise in the VDRL Testdue to the Prozone Effect, or “Hook” Effect, in cases of stronglypositive samples.

The RPR Test is a particle agglutination test. The RPR Test also is amanual test, with a qualitative, subjective, visual readout. The RPRTest utilizes a colloidal suspension of cardiolipin, cholesterol, andlecithin, mixed with micro-particulate carbon. Thus, the RPR Test usesthe same antigen as the VDRL Test, but, in the RPR Test, the antigen hasbeen bound to a carbon particle to allow visualization of theflocculation reaction without the need of a microscope. The mixture isplaced on a test area of a card and the clinician is able to read theresults based upon a visually detectable clumping of the carbonparticles. Like the VDRL Test, the RPR Test is a quantitative test wherethe results require a serial dilution of the test sample and testing ofmultiple dilutions. Thus, the RPR Test is labor intensive and unable tobe automated. Similarly, false negatives arise due to the ProzoneEffect. FIG. 1 is a photograph of a RPR Test card. The clumping of thecarbon particles is shown in a positive test result on the left (circlenumber 9). The right test area of the card (circle number 10), incontrast, where no clumping has occurred, is indicative of a negativeresult.

Three components comprise the VDRL antigen: cardiolipin; cholesterol;and lecithin. These components are lipid in nature. As such, they arenot soluble in aqueous solution. In the classical VDRL test, therefore,the three-component antigen complex is used as a colloidal suspension.It must be prepared under very carefully controlled conditions, justprior to performing the test. Due to its inherent lack of stability, thecolloidal suspension must be prepared daily for immediate use. Becauseof the inherent unstable property, there is no way to preserve the mixedantigens in colloidal suspension for long periods of time. It would bebeneficial to provide a sensitive and specific non-treponemal syphilisscreening and assessment of treatment test, that is stable and has along shelf life.

Neither the VDRL Test nor the RPR Test lend themselves to automation. Itwould be beneficial to provide a syphilis screening and assessment oftreatment test that is able to be automated.

A need exists to overcome the problems with the prior art systems,designs, and processes as discussed above.

SUMMARY OF THE INVENTION

The invention provides serological methods and diagnostic tests forsyphilis antibodies that overcome the hereinafore-mentioneddisadvantages of the heretofore-known devices and methods of thisgeneral type and enables automation and the test has a long shelf life.

The serological methods and diagnostic tests for syphilis antibodiesaccording to the invention combine features of the non-treponemal VDRLTest with the Enzyme Linked Immunosorbent Assay (ELISA) process tocreate an inventive VDRL ELISA Test. ELISA is a test that usesantibodies and color change to identify a substance. It is a type ofanalytic biochemistry assay that uses a solid-phase enzyme immunoassay(EIA) to detect the presence of a substance, usually an antigen or anantibody, in a liquid sample or wet sample.

The steps of ELISA, in this iteration also referred to as “indirect”ELISA, follows the mechanism below:

-   -   A buffered solution of the antigen is added to each well of a        microtiter plate, where it is given time to adhere to the        plastic through charge interactions.    -   A solution of non-reacting protein, such as bovine serum albumin        or casein, is added to block any plastic surface in the well        that remains uncoated by the antigen.    -   The primary antibody is added, which binds specifically to the        test antigen coating the well. This primary antibody could also        be in the serum of a donor to be tested for reactivity towards        the antigen. After a suitable incubation period, the unbound        antibody is decanted and the wells are rinsed with a buffered        solution.    -   A secondary antibody is added, which will bind the primary        antibody. This secondary antibody has an enzyme attached to it,        which has a negligible effect on the binding properties of the        antibody. After a suitable incubation period, the unbound        antibody is decanted and the wells are rinsed with a buffered        solution.    -   A substrate for this enzyme is then added. Often, this substrate        changes color upon reaction with the enzyme. The color change        shows the secondary antibody has bound to primary antibody,        which strongly implies the donor has had an immune reaction to        the test antigen.    -   The higher the concentration of the primary antibody present in        the serum, the stronger the color change. Often, a spectrometer        is used to give quantitative values for color strength.

In ELISA, the enzyme acts as an amplifier; even if only fewenzyme-linked antibodies remain bound, the enzyme molecules will producemany signal molecules. Within common-sense limitations, the enzyme cango on producing color indefinitely, but the more primary antibody ispresent in the donor serum, the more secondary antibody+enzyme willbind, and the faster the color will develop.

ELISA may be run in a qualitative or quantitative format. Qualitativeresults provide a simple positive or negative result (yes or no) for asample. The cutoff between positive and negative is determined by theanalyst and may be statistical. The addition of two or three times thestandard deviation (error inherent in a test) of the mean value obtainedfor known negative samples, is often used to distinguish positive fromnegative samples. In quantitative ELISA, the optical density (OD) of thesample is compared to a standard curve, which is typically a serialdilution of a known-concentration solution of the target molecule. Forexample, if a test sample returns an OD of 1.0, the point on thestandard curve that gave OD=1.0 must be of the same analyteconcentration as the sample.

In sharp contrast to the VDRL and RPR Tests, the VDRL ELISA Test of theinvention, is an enzyme immunoassay, the results of which can be readobjectively with a spectrophotometer. The inventive syphilis test is,therefore, capable of producing standardized, quantitative, andreproducible results, in a single test and to do so in automation. TheVDRL ELISA Test is as sensitive and specific as both the VDRL and RPRtest and is compatible with a wide variety of automated analyzers.

The VDRL and RPR Tests are performed using the antigen complex in theform of a colloidal suspension. In contrast, the antigen complex used inthe inventive VDRL ELISA Test is prepared by dissolving the three lipidcomponents in a mixture of organic solvents. This produces a uniform,homogeneous antigen that can be accurately dispensed into microwells,and dried in place. This results in VDRL antigen wells that areuniformly coated. The uniformity of coating facilitates accurate andreproducible test results. The VDRL ELISA antigen coated wells may bestored for extended periods then used to perform an ELISA test.Heretofore, it has been generally understood that wells coated with theVDRL antigen complex could not be stored for extended periods of timeand, after such storage, be used to perform in an ELISA test in anaccurate and reproducible manner.

The exemplary embodiments described herein utilize microwells, forexample, polystyrene microwells, as the solid-phase of the VDRL ELISAtest. Use of the term microwells is not to be taken as limiting anyplatform of any embodiment only to such microwells. This term, as usedanywhere herein, is defined as equally including all other solid-phaseplatforms that can be substituted for microwells, which platforms aredefined to include, but are not limited to, microtubes, columns, beads,dipsticks, nitrocellulose membranes, lateral flow devices, and othercontainers, to name a few. As such, the solid-phase platforms can alsobe described in general as containers.

The design of the VDRL ELISA Test is significantly improved over theprior art and permits quantization and standardization of the testresults, as follows:

-   -   1) A kit calibrator facilitates standardization and        normalization of the test results. The calibrator(s) is a        standard serum sample that is traceable to a primary, or        secondary, standard, and has been assigned a known value. The        calibrator(s) is included with the patient samples in each test        run to produce a result of known value against which patients        test results may be compared and thereby interpreted as either        positive or negative.    -   2) The kit's negative and positive controls facilitate the        validation of the test results. The positive and negative        controls are serum samples that have been assigned expected        value ranges and that are included with the patient samples in        each test run. If the values obtained for the positive and        negative controls fall within their assigned ranges when        compared to the kit calibrator, the results obtained for the        patient samples are valid, if the values do not fall within the        assigned ranges, the results obtained for the patient samples        are invalid and the test must be repeated.

The formulations contributing to the sensitivity and specificity of theVDRL ELISA Test include the following:

-   -   1) The formulation, i.e., relative concentrations of the        cardiolipin, cholesterol, lecithin antigen preparation,        contributes to the sensitivity and specificity of the test.    -   2) The formulation of a post-coating solution prevents        non-specific binding of human antibody to the antigen-coated        wells.    -   3) The formulation of a sample diluent prevents the non-specific        binding of human antibody to the antigen-coated wells. The        sample diluent is a buffered solution that contains an inert        protein that does not interfere with the binding of the patient        antibody that is specifically directed against the antigen        coated on the wells. Concurrently, the inert protein prevents        the binding of extraneous patient antibody that is not        specifically directed against the antigen coated on the wells,        thereby preventing potentially false positive results.    -   4) In cases of very high levels of non-treponemal antibody, the        VDRL ELISA Test is not subject to the Prozone or Hook Effect,        which can lead to false negative results in the VDRL and RPR        Tests.

The properties of the VDRL antigen mixture that have heretoforeprevented the development of a stable and reproducible VDRL ELISA test,are as follows:

1) Cardiolipin, a major component of the VDRL antigen preparation, isinherently unstable.

-   -   2) Cardiolipin and the other components of the antigen        preparation, cholesterol and lecithin, are not soluble in        aqueous solution and, therefore, cannot be bound to polystyrene        wells by conventional measures.

The inventive systems and processes are not limited to particularcardiolipins or cholesterol. The inventive systems and processes areenvisioned to use natural or purified-from-natural cardiolipins or evensynthesized cardiolipins. It is possible that synthesized cardiolipinscould be used even if they are inherently stable. The inventive systemsand processes are envisioned to also be used with synthesizedcholesterol and/or synthesized lecithin.

The following procedures were used to prepare VDRL antigen coated wellsthat are stable and capable of producing reproducible test results:

1) The cholesterol is initially dissolved in an organic solvent, and isthen further diluted in an ethanol solution containing cardiolipin andlecithin.

-   -   2) A small volume (e.g., 50 microliter) of the antigen solution        is permitted to evaporate in place, in polystyrene microwells.    -   3) The coated wells are rinsed once with buffered saline.    -   4) The antigen coating is then stabilized by overcoating with an        inert protein dissolved in buffered saline.    -   5) The overcoat solution is decanted and the wells are air-dried        and then sealed in vapor-proof pouches with desiccant.    -   6) The enzyme-labeled conjugate component of the test is        formulated to be compatible with the lipid nature of the VDRL        antigens coated on the wells.    -   7) The sample diluent is formulated to be compatible with the        lipid nature of the VDRL antigens coated on the wells.    -   8) The wash fluid is formulated to be compatible with the lipid        nature of the VDRL antigens coated on the wells.    -   9) The enzyme substrate is formulated to be compatible with the        lipid nature of the VDRL antigens coated on the wells.

With the foregoing and other objects in view, there is provided, inaccordance with the invention, a method of manufacturing anon-treponemal diagnostic test for syphilis infection includes the stepsof initially dissolving cholesterol in an organic solvent and furtherdiluting the dissolved cholesterol in an ethanol solution comprisingcardiolipin and lecithin to form an antigen solution, permitting avolume of the antigen solution to evaporate in place within a containerand at least partially coat the container with an antigen coating, andstabilizing the antigen coating into a syphilis antigen complex byovercoating the antigen coating with an overcoat solution comprising aninert protein.

With the objects of the invention in view, there is also provided amethod of manufacturing a non-treponemal diagnostic test for syphilisinfection including the steps of initially dissolving cholesterol in anorganic solvent and further diluting the dissolved cholesterol in anethanol solution comprising cardiolipin and lecithin to form an antigensolution, permitting a volume of the antigen solution to evaporate inplace within a container and at least partially coat the container withan antigen coating, stabilizing the antigen coating into a syphilisantigen complex by overcoating the antigen coating with an overcoatsolution comprising an inert protein, providing an enzyme-labeledconjugate component of a syphilis infection test that is formulated tobe compatible with a lipid nature of the cholesterol, the cardiolipin,and the lecithin, providing a sample diluent that is formulated to becompatible with the lipid nature of the cholesterol, the cardiolipin,and the lecithin, providing a wash fluid that is formulated to becompatible with the lipid nature of the cholesterol, the cardiolipin,and the lecithin, and providing an enzyme substrate that is formulatedto be compatible with the lipid nature of the cholesterol, thecardiolipin, and the lecithin.

In accordance with another mode of the invention, there are alsoprovided the steps of rinsing the at least partially coated containerwith buffered saline and decanting the overcoat solution, drying thecontainer, and sealing the container in a vapor-proof pouch.

In accordance with a further mode of the invention, there are alsoprovided the steps of, after the step of permitting a volume of theantigen solution to evaporate, rinsing the at least partially coatedcontainer with buffered saline, and, after the step of stabilizing theantigen coating into a syphilis antigen complex, decanting the overcoatsolution, drying the container, and sealing the container in avapor-proof pouch.

In accordance with an added mode of the invention, the volume of theantigen solution is approximately 50 microliters.

In accordance with an additional mode of the invention, the container isone or more of a microwell, a polystyrene microwell, a microtube, acolumn, a bead, a dipstick, a nitrocellulose membrane, and a lateralflow device.

In accordance with yet another mode of the invention, the organicsolvent is one or more of acetone, chloroform, butanol, methanol, orether.

In accordance with yet a further mode of the invention, the cholesterolis a natural cholesterol, a purified-from-natural cholesterol, and/or asynthesized cholesterol.

In accordance with yet an added mode of the invention, the cardiolipinis a natural cardiolipin, a purified-from-natural cardiolipin, and/or asynthesized cardiolipin.

In accordance with yet an additional mode of the invention, the lecithinis a natural lecithin, a purified-from-natural lecithin, and/or asynthesized lecithin.

In accordance with again another mode of the invention, the container isair-dried.

In accordance with again a further mode of the invention, there isprovided the step of sealing the container in the vapor-proof pouch withdesiccant.

In accordance with again an added mode of the invention, the overcoatingstep is carried out with the inert protein selected from one or more ofbovine serum, bovine serum albumin, fetal bovine serum, gelatin, goatserum, horse serum, and milk protein.

In accordance with again an additional mode of the invention, theovercoating step is carried out by dissolving the inert protein inbuffered saline.

In accordance with still another mode of the invention, the overcoatingstep is carried out by leaving the saline solution of the inert proteinin the at least partially coated container for a period of timesufficient to allow the inert protein to bind to non-coated portions ofthe container.

In accordance with still a further mode of the invention, the period oftime is at least one of between approximately 30 minutes andapproximately 5 hours, between approximately 1 and approximately 3hours, between approximately 1.5 and approximately 2.5 hours, orapproximately 2 hours.

In accordance with still an added mode of the invention, the stabilizedantigen coating overcoated with the inert protein has a shelf life ofbetween approximately 6 months and approximately 1 year.

In accordance with still an additional mode of the invention, anenzyme-labeled conjugate component of a syphilis infection test that isformulated to be compatible with a lipid nature of the cholesterol, thecardiolipin, and the lecithin is provided, a sample diluent that isformulated to be compatible with the lipid nature of the cholesterol,the cardiolipin, and the lecithin is provided, a wash fluid that isformulated to be compatible with the lipid nature of the cholesterol,the cardiolipin, and the lecithin is provided, and an enzyme substratethat is formulated to be compatible with the lipid nature of thecholesterol, the cardiolipin, and the lecithin is provided.

In accordance with another mode of the invention, there are provided thesteps of adding serum of a donor to be tested for reactivity towards thesyphilis antigen complex into the container and allowing the syphilisantibody to bind to the antigen coating if the syphilis antibody existsin the patient's serum, rinsing the container with buffered saline,adding a secondary antibody designed to bind to the primary antibody inthe serum to the container, the secondary antibody having an attachedenzyme that changes color when the enzyme substrate for the enzyme isadded thereto and reacts with the enzyme, rinsing the container withbuffered saline, and adding the enzyme substrate to produce a colorchange if the serum contains the syphilis antibody.

In accordance with another mode of the invention, there is provided thestep of examining contents of the container with an automatedspectrophotometric analyzer to detect the color change of the testedserum and, if there is a color change showing that the secondaryantibody has bound to the primary antibody, forming a test resultconcluding that the donor of the serum has had an immune reaction to thetest syphilis antigen.

In accordance with a concomitant mode of the invention, there isprovided the step of producing with the spectrophotometer quantitativevalues corresponding to a strength of the color change.

Although the invention is illustrated and described herein as embodiedin serological methods and diagnostic tests for syphilis antibodies, itis, nevertheless, not intended to be limited to the details shownbecause various modifications and structural changes may be made thereinwithout departing from the spirit of the invention and within the scopeand range of equivalents of the claims. Additionally, well-knownelements of exemplary embodiments of the invention will not be describedin detail or will be omitted so as not to obscure the relevant detailsof the invention.

Additional advantages and other features characteristic of the presentinvention will be set forth in the detailed description that follows andmay be apparent from the detailed description or may be learned bypractice of exemplary embodiments of the invention. Still otheradvantages of the invention may be realized by any of theinstrumentalities, methods, or combinations particularly pointed out inthe claims.

Other features that are considered as characteristic for the inventionare set forth in the appended claims. As required, detailed embodimentsof the present invention are disclosed herein; however, it is to beunderstood that the disclosed embodiments are merely exemplary of theinvention, which can be embodied in various forms. Therefore, specificstructural and functional details disclosed herein are not to beinterpreted as limiting, but merely as a basis for the claims and as arepresentative basis for teaching one of ordinary skill in the art tovariously employ the present invention in virtually any appropriatelydetailed structure. Further, the terms and phrases used herein are notintended to be limiting; but rather, to provide an understandabledescription of the invention. While the specification concludes withclaims defining the features of the invention that are regarded asnovel, it is believed that the invention will be better understood froma consideration of the following description in conjunction with thedrawing figures, in which like reference numerals are carried forward.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, where like reference numerals refer toidentical or functionally similar elements throughout the separateviews, which are not true to scale, and which, together with thedetailed description below, are incorporated in and form part of thespecification, serve to illustrate further various embodiments and toexplain various principles and advantages all in accordance with thepresent invention. Advantages of embodiments of the present inventionwill be apparent from the following detailed description of theexemplary embodiments thereof, which description should be considered inconjunction with the accompanying drawings in which:

FIG. 1 is a photograph of a portion of an RPR Test card showing one testarea with a positive result and one test area with a negative result;

FIG. 2 is a process flow diagram for manufacturing an exemplaryembodiment of a VDRL ELISA Test;

FIG. 3 is a process flow diagram for conducting the VDRL ELISA Test;

FIG. 4 is a diagram illustrating the reaction in the VDRL ELISA Test;and

FIG. 5 is a graph illustrating a comparison of results between the VDRLELISA Test and the RPR Test and demonstrating a prozone or “hook” effectin the RPR test.

DETAILED DESCRIPTION OF THE INVENTION

As required, detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely exemplary of the invention, which can be embodied in variousforms. Therefore, specific structural and functional details disclosedherein are not to be interpreted as limiting, but merely as a basis forthe claims and as a representative basis for teaching one skilled in theart to variously employ the present invention in virtually anyappropriately detailed structure. Further, the terms and phrases usedherein are not intended to be limiting; but rather, to provide anunderstandable description of the invention. While the specificationconcludes with claims defining the features of the invention that areregarded as novel, it is believed that the invention will be betterunderstood from a consideration of the following description inconjunction with the drawing figures, in which like reference numeralsare carried forward.

DEFINITIONS

Before the present invention is disclosed and described, it is to beunderstood that the terminology herein is not intended to be limitingand is only being used for the purpose of describing particularembodiments. Unless otherwise specified, all technological terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the present invention belongs.

Alternate embodiments may be devised without departing from the spiritor the scope of the invention. Additionally, well-known elements ofexemplary embodiments of the invention will not be described in detailor will be omitted so as not to obscure the relevant details of theinvention.

As used herein, the terms “a” or “an”, are defined as one or more thanone. The term “plurality”, as used herein, is defined as two or morethan two. The term “another”, as used herein, is defined as at least asecond or more.

As used herein, the terms “including” and/or “having”, are defined ascomprising (i.e., open language). The terms “comprises,” “comprising,”or any other variation thereof are intended to cover a non-exclusiveinclusion, such that a process, method, article, or apparatus thatcomprises a list of elements does not include only those elements butmay include other elements not expressly listed or inherent to suchprocess, method, article, or apparatus. An element proceeded by“comprises . . . a” does not, without more constraints, preclude theexistence of additional identical elements in the process, method,article, or apparatus that comprises the element.

As used herein, relational terms, such as first and second, may be usedsolely to distinguish one entity or action from another entity or actionwithout necessarily requiring or implying any actual such relationshipor order between such entities or actions.

As used herein, the term “coupled” is defined as connected, although notnecessarily directly, and not necessarily mechanically.

As used herein, the term “about” or “approximately” applies to allnumeric values, whether or not explicitly indicated. These termsgenerally refer to a range of numbers that one of skill in the art wouldconsider equivalent to the recited values (i.e., having the samefunction or result). In many instances these terms may include numbersthat are rounded to the nearest significant figure.

Herein various embodiments of the present invention are described. Inmany of the different embodiments, features are similar. Therefore, toavoid redundancy, repetitive description of these similar features maynot be made in some circumstances. It shall be understood, however, thatdescription of a first-appearing feature applies to the later describedsimilar feature and each respective description, therefore, is to beincorporated therein without such repetition.

Described now are exemplary embodiments of the present invention.Referring now to the figures of the drawings in detail and first,particularly to FIG. 2, there is shown a first exemplary embodiment of aprocess for creating and manufacturing a VDRL ELISA Test. The inventiveVDRL ELISA Test starts with the previous understanding that the antigencomplex for syphilis is formed from a combination of cardiolipin,cholesterol and lecithin, which, due to its lipid nature, does notreadily remain in a microwell. From this, it was necessary to find a wayto obtain a deposition of the antigen complex in a microwell that notonly remains for periods of time sufficient to perform a non-treponemaltest for syphilis, but also to perform that test with microwells havinga desirably long shelf life.

The process for creating such a microwell according to the invention isillustrated with the flow chart of FIG. 2. It is noted that, asdescribed above, use of the term “microwells” in the exemplaryembodiments is not to be taken as limiting the platform only tomicrowells and equally includes all other solid-phase platforms can besubstituted for microwells, which can include microtubes, columns,beads, dipsticks, nitrocellulose membranes, and lateral flow devices.

As set forth above, the non-binding of the antigen complex to amicrowell is due the lipid nature of cardiolipin, cholesterol andlecithin, which compounds are not soluble in aqueous solution. Theinventors discovered, however, that these compounds can be dissolved inorganic solvents and dried in wells without a deleterious affect uponthe compounds. The inventors have further discovered that the relativeproportions of the compounds used in the classical VDRL method, whendiluted to one-tenth the concentration, i.e., 0.003% W/V cardiolipin,0.09% W/V cholesterol and 0.02% W/V lecithin, produce the desiredresults when dried in wells in the VDRL-ELISA method. Furthermore, byexamining the aforementioned compounds in the VDRL ELISA method, eitherseparately or in various relative proportions, the inventors determinedthat the relative concentrations stated above are more reactive withnon-treponemal syphilis antibodies than any of the compoundsindividually or in any of the other relative proportions examined.Accordingly, in Step 210, cholesterol is dissolved in an organicsolvent, such as acetone, chloroform, butanol, methanol, and/or ether.In Step 220, cardiolipin and lecithin are dissolved in ethanol, and theorganic solvent mixture is further diluted in the ethanol solutioncontaining the cardiolipin and lecithin. In Step 230, the two mixturesare combined and an amount of this mixture is placed in microwells, forexample, polystyrene microwells. In an exemplary embodiment, the amountof the mixture placed in each microwell is between approximately 40 and60 microliters, in particular, approximately 50 microliters.

It is known that both the organic solvent and ethanol evaporate readily.From this, evaporation of the organic solvent and ethanol is allowed tooccur in Step 240, which evaporation can be aided with air circulationdevices such as fans. In an exemplary embodiment, the evaporation occursat room temperature (20° C. to 25° C.). Evaporation continues until theliquid in the microwells has dried. At this point, the three componentsof the antigen complex become bound to the interior surface of themicrowells. The microwells are washed once, for example, with bufferedsaline in Step 250.

After the microwells are coated with the antigen complex, it was foundthat this coating is not present on the entirety of the microwellinterior. Where such uncoated portions occur, it is possible that, whenused in the actual testing procedure, the test material (i.e., thepatient's serum potentially containing the syphilis antibody) could bindor be adversely affected by the interior surface of the microwell. Insuch a case, the test material could bond to the non-coated surfaces andundesirably augment the result, which occurs, as set forth above, byspectrophotometric reading. In such a situation, the color reading willbe more intense than it should be for that test specimen. Therefore, itwould be desirable to prevent this detrimental situation from occurring.To solve this problem, the inventive manufacturing process binds anon-reactive compound to the uncoated portions of the microwells. Theselected non-reactive compound is an inert protein, such as any one ofbovine serum, bovine serum albumin, fetal bovine serum, gelatin, goatserum, horse serum, and milk protein, to name a few. Utilizing the knownproperty that cardiolipin, cholesterol, and lecithin are not soluble inaqueous solution, in Step 260, the coated microwells are filled with apost-coating mixture of the inert protein dissolved in buffered saline.This aqueous solution is left in the microwells (not adversely affectingthe coating of the antigen complex) in Step 270 for a given period oftime sufficient to allow the inert protein to bind to the non-coatedportions of the microwell. This post-coating soaking occurs for betweenapproximately 30 minutes and approximately 5 hours, for example, betweenapproximately 1 and approximately 3 hours, between approximately 1.5 andapproximately 2.5 hours, and, in particular, for approximately 2 hours,resulting in a bond of the inert protein in all areas where the antigencomplex does not appear. Removal of the inert protein soak occurs inStep 280 by decanting or aspirating the remaining solution. Themicrowell interiors are air dried and then sealed in vapor-proof poucheswith desiccant in Step 290. When the so-coated microwells are packagedwith desiccant, it was discovered that the antigen complex and thepost-coating inert protein remained stable for many months, whichstability was found to be augmented by the non-specific binding of theinert protein. In such a packed state, the shelf-life of the so-coatedinterior is at least 12 months. In particular, the shelf-life is between6 months and 1 year.

With these coated microwells, the steps of the VDRL ELISA Test followthe mechanism below and is illustrated with reference to the flow chartof FIG. 3 and the diagram of FIG. 4. In Step 310, the serum 410 of adonor to be tested for reactivity towards the syphilis antigen complex420 is added into one or more microwells in a microtiter plate. Becausethe syphilis antigen complex 420 coats each microwell, if the syphilisantibody exists in the patient's serum 410 binds, that antibody willbind to the coating 420 in Step 320. The microwell is rinsed, forexample, with buffered saline in Step 330. A secondary antibody 430 isadded in Step 340, which antibody 430 is designed to bind to the primaryantibody in the serum 410, and the microwell is rinsed, for example,with buffered saline in Step 350. This secondary antibody 430 has anenzyme E attached to it, which has a negligible effect on the bindingproperties of the antibody. However, when a substrate for the enzyme Eis added, the substrate will change color upon reaction with the enzymeE. This substrate is added in Step 360 to produce a color change in Step370 if the serum 410 contains the syphilis antibody. An automatedspectrophotometric analyzer examines the reactions in Step 380 to detectthe color change of the tested serum 410. Any color change in themicrowell shows that the secondary antibody 430 has bound to primaryantibody 410, which strongly implies that the donor of the serum 410 hashad an immune reaction to the test syphilis antigen. The higher theconcentration of the primary syphilis antibody present in the serum 410,the stronger the color change will be in Step 370. The spectrophotometerproduces, in Step 390, quantitative values for the color strength. Asthose having skill in the art know, this test entirely eliminates theProzone Effect, which means, in practice, that a lab does not need toperform further testing where the sample is diluted and the sample isre-tested, perhaps repeatedly. The difference between the results of theVDRL ELISA Test and the tests that have the Prozone Effect is shown inthe graph of FIG. 5, where a plateau occurs in the inventive test withlater titration demonstrating a decreasing the dose effect. Moreover,this test is able to be readily and easily automated by standardautomated spectrophotometric analyzers. It has been found that thesensitivity is at least as equivalent to the RPR and VDRL Tests but thelong shelf life and ability to be automated (thereby eliminating theneed for subjective analysis by a clinician).

The classical non-treponemal tests, VDRL and RPR, are known to detectboth IgG and IgM class antibodies, but they not capable ofdistinguishing between the two antibody classes. The inventors are awarethat the VDRL ELISA Test can be modified based upon the specificity ofthe enzyme-labeled conjugate being used in the test. Therefore, it ispossible to detect IgG only, IgM only, or both. In one iteration, theVDRL test can be used for screening for the presence of non-treponemalsyphilis antibodies in adults by employing conjugates that detect bothIgG and IgM. This is the approach recommended by the Centers for DiseaseControl (CDC) for syphilis screening. In another iteration, the VDRLELISA Test can be modified to detect only IgM antibodies. It is knownthat IgM antibodies of maternal origin do not cross the placentalbarrier, therefore, IgM antibodies to syphilis detected in the newborn'sserum is an indication of syphilis infection in the newborn. It followsthat this iteration of the VDRL ELISA Test can be applied to thediagnosis of syphilis in the newborn in a novel exemplary embodiment ofthe VDRL ELISA Test.

The foregoing description and accompanying drawings illustrate theprinciples, exemplary embodiments, and modes of operation of theinvention. However, the invention should not be construed as beinglimited to the particular embodiments discussed above. Additionalvariations of the embodiments discussed above will be appreciated bythose skilled in the art and the above-described embodiments should beregarded as illustrative rather than restrictive. Accordingly, it shouldbe appreciated that variations to those embodiments can be made by thoseskilled in the art without departing from the scope of the invention asdefined by the following claims.

What is claimed is:
 1. A method of detecting antibodies to syphilisantigens, which comprises: providing a syphilis detection test having: ascreening solid-phase platform having a screening portion coated withnon-treponemal syphilis antigens, the screening portion being fabricatedby: initially dissolving cholesterol in an organic solvent and furtherdiluting the dissolved cholesterol in an ethanol solution comprisingcardiolipin and lecithin to form an antigen solution; permitting avolume of the antigen solution to evaporate in place at the screeningportion and at least partially coating the screening portion with anantigen coating; and stabilizing the antigen coating into a syphilisantigen complex by overcoating the antigen coating with an overcoatsolution comprising an inert protein; and a confirmatory solid-phaseplatform having a confirmation portion coated with treponemal syphilisantigens; running a syphilis detection test on the screening portion andrunning the syphilis detection test on the confirmation portion; andconfirming a true positive if syphilis antibodies are detected at boththe screening portion and the confirmation portion.
 2. The methodaccording to claim 1, wherein the solid-phase platforms are selectedfrom at least one of the group of microwells, microtubes, columns,beads, dipsticks, nitrocellulose membranes, and lateral flow devices. 3.The method according to claim 1, wherein the screening portion isfabricated by: rinsing the at least partially coated screening portionwith buffered saline; and decanting the overcoat solution, drying thescreening portion, and sealing the screening portion in a vapor-proofpouch.
 4. The method according to claim 1, which further comprises:after the step of permitting a volume of the antigen solution toevaporate, rinsing the at least partially coated screening portion withbuffered saline; and after the step of stabilizing the antigen coatinginto a syphilis antigen complex, decanting the overcoat solution, dryingthe screening portion, and sealing the screening portion in avapor-proof pouch.
 5. The method according to claim 1, wherein thevolume of the antigen solution is approximately 50 microliters.
 6. Themethod according to claim 1, wherein the organic solvent is one or moreof acetone, chloroform, butanol, methanol, or ether.
 7. The methodaccording to claim 1, wherein the cholesterol is a natural cholesterol,a purified-from-natural cholesterol, and/or a synthesized cholesterol.8. The method according to claim 1, wherein the cardiolipin is a naturalcardiolipin, a purified-from-natural cardiolipin, and/or a synthesizedcardiolipin.
 9. The method according to claim 1, wherein the lecithin isa natural lecithin, a purified-from-natural lecithin, and/or asynthesized lecithin.
 10. The method according to claim 3, wherein thescreening portion is fabricated by air-drying the screening portion. 11.The method according to claim 3, which further comprises sealing thescreening portion in the vapor-proof pouch with desiccant.
 12. Themethod according to claim 1, which further comprises carrying out theovercoating step with the inert protein selected from one or more ofbovine serum, bovine serum albumin, fetal bovine serum, gelatin, goatserum, horse serum, and milk protein.
 13. The method according to claim1, which further comprises carrying out the overcoating step bydissolving the inert protein in buffered saline.
 14. The methodaccording to claim 13, which further comprises carrying out theovercoating step by leaving the saline solution of the inert protein inthe at least partially coated screening portion for a period of timesufficient to allow the inert protein to bind to non-coated portions ofthe screening portion.
 15. The method according to claim 14, wherein theperiod of time is at least one of: between approximately 30 minutes andapproximately 5 hours; between approximately 1 and approximately 3hours; between approximately 1.5 and approximately 2.5 hours; orapproximately 2 hours.
 16. The method according to claim 1, wherein thestabilized antigen coating overcoated with the inert protein has a shelflife of between approximately 6 months and approximately 1 year.
 17. Themethod according to claim 3, which further comprises: providing anenzyme-labeled conjugate component of a syphilis infection test that isformulated to be compatible with a lipid nature of the cholesterol, thecardiolipin, and the lecithin; providing a sample diluent that isformulated to be compatible with the lipid nature of the cholesterol,the cardiolipin, and the lecithin; providing a wash fluid that isformulated to be compatible with the lipid nature of the cholesterol,the cardiolipin, and the lecithin; and providing an enzyme substratethat is formulated to be compatible with the lipid nature of thecholesterol, the cardiolipin, and the lecithin.
 18. The method accordingto claim 17, which further comprises: adding serum of a donor to betested for reactivity towards the syphilis antigen complex into thescreening portion and allowing the syphilis antibody to bind to theantigen coating if the syphilis antibody exists in the patient's serum;rinsing the screening portion with buffered saline; adding a secondaryantibody designed to bind to the primary antibody in the serum to thescreening portion, the secondary antibody having an attached enzyme thatchanges color when the enzyme substrate for the enzyme is added theretoand reacts with the enzyme; rinsing the screening portion with bufferedsaline; and adding the enzyme substrate to produce a color change if theserum contains the syphilis antibody.
 19. The method according to claim18, which further comprises examining contents of the screening portionwith an automated spectrophotometric analyzer to detect the color changeof the tested serum and, if there is a color change showing that thesecondary antibody has bound to the primary antibody, forming a testresult concluding that the donor of the serum has had an immune reactionto the test syphilis antigen.
 20. The method according to claim 19,which further comprises producing with the spectrophotometerquantitative values corresponding to a strength of the color change.